WO2019218898A1 - Crosslinked protein-based separation membrane and application thereof - Google Patents

Crosslinked protein-based separation membrane and application thereof Download PDF

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Publication number
WO2019218898A1
WO2019218898A1 PCT/CN2019/085848 CN2019085848W WO2019218898A1 WO 2019218898 A1 WO2019218898 A1 WO 2019218898A1 CN 2019085848 W CN2019085848 W CN 2019085848W WO 2019218898 A1 WO2019218898 A1 WO 2019218898A1
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protein
membrane
separation membrane
crosslinked
based separation
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PCT/CN2019/085848
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French (fr)
Chinese (zh)
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杨鹏
杨发翠
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陕西师范大学
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Priority to US17/263,264 priority Critical patent/US20210187448A1/en
Publication of WO2019218898A1 publication Critical patent/WO2019218898A1/en
Priority to US17/579,209 priority patent/US12011694B2/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/44—Polymers obtained by reactions only involving carbon-to-carbon unsaturated bonds, not provided for in a single one of groups B01D71/26-B01D71/42
    • B01D71/441—Polyvinylpyrrolidone
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/24—Dialysis ; Membrane extraction
    • B01D61/243—Dialysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0002—Organic membrane manufacture
    • B01D67/0006—Organic membrane manufacture by chemical reactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0081—After-treatment of organic or inorganic membranes
    • B01D67/0093—Chemical modification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0081—After-treatment of organic or inorganic membranes
    • B01D67/0093—Chemical modification
    • B01D67/00931—Chemical modification by introduction of specific groups after membrane formation, e.g. by grafting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/10—Supported membranes; Membrane supports
    • B01D69/107—Organic support material
    • B01D69/1071—Woven, non-woven or net mesh
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/12—Composite membranes; Ultra-thin membranes
    • B01D69/125—In situ manufacturing by polymerisation, polycondensation, cross-linking or chemical reaction
    • B01D69/1251—In situ manufacturing by polymerisation, polycondensation, cross-linking or chemical reaction by interfacial polymerisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/142—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers"
    • B01D69/144—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers" containing embedded or bound biomolecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/48—Polyesters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/30—Cross-linking
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/38—Graft polymerization
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/40—Details relating to membrane preparation in-situ membrane formation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/02—Details relating to pores or porosity of the membranes
    • B01D2325/0283—Pore size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/04—Characteristic thickness
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0002—Organic membrane manufacture
    • B01D67/0023—Organic membrane manufacture by inducing porosity into non porous precursor membranes
    • B01D67/0032—Organic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods
    • B01D67/0034—Organic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods by micromachining techniques, e.g. using masking and etching steps, photolithography

Definitions

  • the present invention relates to a separation membrane, and in particular to a separation membrane which is simple to prepare, green, mild and controllable in pore size, and the use of the separation membrane.
  • Membrane separation technology has been widely used in petrochemical, sewage treatment, medical and health, food processing and other fields due to its high separation efficiency, no secondary pollution and simple operation.
  • Membrane material is one of the core contents of membrane separation technology.
  • the commonly used membrane materials are mainly organic synthetic polymer materials.
  • the preparation method of polymer membrane is complicated, and it is easy to adsorb organic substances (such as proteins, colloids and microorganisms in the raw material liquid).
  • non-specific adsorption of proteins on the surface of the membrane may cause negative effects such as blood coagulation, resulting in a decrease in the amount of permeation of the separation membrane and a decrease in the specificity of separation.
  • Hemodialysis treatment is one of the commonly used treatments for uremic patients.
  • the blood purification membrane mainly exchanges substances through diffusion/convection principle, and achieves the purpose of eliminating metabolic wastes in the body, maintaining electrolytes and removing excess water from the body.
  • Conventional hemodialysis membranes are unable to achieve the elimination of macromolecular toxins. Therefore, it is of great practical significance to develop a separation membrane which has good separation performance and is simple and inexpensive, and is inexpensive.
  • the technical problem to be solved by the present invention is to provide a separation membrane based on crosslinked proteins and the use of the separation membrane in view of the above-mentioned drawbacks of the prior art.
  • the cross-linked protein-based separation membrane used to solve the above technical problem is: a protein two-dimensional nano-film formed by phase-transformation of a protein is cross-linked with a crosslinking agent and adhered to the porous membrane to form a cross-linked protein nano-film as a dense
  • the cortex and the porous membrane are separation membranes of the support layer.
  • the above protein is any one of lysozyme, bovine serum albumin, insulin, and ⁇ -lactalbumin.
  • the lysozyme phase is transformed into a two-dimensional nanofilm of lysozyme: buffering 4-hydroxyethylpiperazineethanesulfonic acid of 10-100 mmol/L tris(2-carboxyethyl)phosphine
  • the solution is adjusted to pH 6.0-8.0 with NaOH, and then mixed with 1 to 30 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution in equal volume and directly spread on the surface of the substrate, and cultured at room temperature 2 ⁇ 6 hours, a layer of lysozyme two-dimensional nano-film was formed on the gas-liquid interface of the liquid on the substrate.
  • the two-dimensional nanofilm formed by protein phase transformation is: 10 to 100 mmol/L of tris(2-carboxyethyl)phosphine
  • the 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution was adjusted to a pH of 4.0 to 6.0 with NaOH, and then mixed with an equal volume of 1-hydroxyethylpiperazineethanesulfonic acid buffer solution of 1 to 30 mg/mL protein.
  • the surface of the substrate is directly spread and incubated at room temperature for 2 to 6 hours to form a layer of protein two-dimensional nano-film on the liquid-liquid interface of the liquid on the substrate.
  • the protein two-dimensional nano film formed by the phase transition of the above protein is crosslinked by a crosslinking agent: the protein two-dimensional nano film is transferred to an aqueous solution of a crosslinking agent having a mass fraction of 0.2% to 2%, and crosslinked at room temperature by 2 to 6 hour.
  • the crosslinking agent is any one of glutaraldehyde, genipin, glutamine transaminase, and carbodiimide.
  • the porous film described above is any one of a PET nucleus membrane, a PC membrane, a PP membrane, a PSF membrane, and a PTFE membrane, and the pore size of the porous membrane is 0.22 to 10 ⁇ m.
  • the separation membrane based on the crosslinked protein of the present invention can be used as a dialysis membrane or an ultrafiltration membrane.
  • the present invention is based on the use of a separation membrane of crosslinked proteins as a dialysis membrane for the separation of mixed proteins such as bovine serum albumin and insulin, or myoglobin and insulin.
  • the present invention is based on the use of a crosslinked protein separation membrane as a dialysis membrane for the separation of mixed dyes such as methyl blue and methyl orange, or methyl blue and rhodamine B.
  • the invention is based on the use of a separation membrane of crosslinked protein as a dialysis membrane for removing urinary toxins from hemodialysis, wherein the urinary toxin is a toxin having a relative molecular weight of ⁇ 500, such as urea, creatine, etc., and a relative molecular weight of 500 ⁇ .
  • Medium molecular weight toxins of 20000 such as ⁇ -microglobulin, parathyroid hormone, leptin, renin, protein-binding toxin, sulphuric acid, and the like.
  • the separation membrane of the present invention is densely packed from oligomers of proteins, is colorless and transparent, and is a pure protein membrane.
  • the thickness of the membrane can be controlled according to the concentration of the protein, and the pore size decreases from 3.4 nm as the thickness of the membrane increases. To 1.8nm.
  • the separation membrane of the invention has good biocompatibility, can be used as a dialysis membrane for blood purification, can remove urea, creatine liver and medium macrotoxin and sulphuric acid phenol, and has more for macromolecular proteins. High rejection rate is an ideal dialysis membrane material.
  • the separation membrane of the present invention is capable of efficiently separating mixed proteins of different sizes and mixed dyes of different sizes, such as bovine serum albumin and insulin, myoglobin and insulin, methyl blue and methyl orange, methyl blue and rhodamine B.
  • the preparation method of the separation membrane of the invention is simple, easy to realize large-area preparation, and has the characteristics of low cost, low energy consumption, environmental protection, etc., avoiding the cumbersome steps and environmental pollution problems in the traditional polymer membrane synthesis process.
  • Example 1 is a scanning electron micrograph of a crosslinked lysozyme nanofilm of Example 1.
  • Example 2 is a transmission electron microscope plan view of the crosslinked lysozyme nanofilm of Example 1.
  • Example 3 is an atomic force microscope chart of the crosslinked lysozyme nanofilm of Example 1.
  • Figure 4 is a pore size distribution of crosslinked lysozyme nanofilms of different thicknesses.
  • Fig. 5 is a view showing the removal of urea, sulphate, creatine and ⁇ -microglobulin by the dialysis membrane obtained in Example 1.
  • 60 ⁇ L of 50 mmol/L tris(2-carboxyethyl)phosphine 4-hydroxyethylpiperazineethanesulfonic acid buffer solution was adjusted to pH 7.0 with NaOH, and then combined with 60 ⁇ L of 2 mg/mL lysozyme 4-hydroxyl
  • the ethyl piperazine ethanesulfonic acid buffer solution was uniformly mixed and directly spread on the surface of the 18 mm ⁇ 18 mm coverslip, and incubated at room temperature for 2 hours to form a layer of lysozyme two-dimensional nano-film on the liquid-liquid interface of the cover glass.
  • the lysozyme two-dimensional nanofilm at the gas-liquid interface was transferred to an aqueous solution of glutaraldehyde having a mass fraction of 1%, and after cross-linking at room temperature for 2 hours, a cross-linked lysozyme nanofilm was obtained.
  • the cross-linked lysozyme nano-film was adhered to a PET nuclear pore membrane with a membrane diameter of 25 mm, a thickness of 12 ⁇ m and a pore size of 10 ⁇ m, and a cross-linked lysozyme nanofilm was used as a dense skin layer and a PET nucleus membrane as a support layer. Dialysis membrane.
  • Example 2 the 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 4 mg/mL lysozyme 4-hydroxyethylpiperazineethanesulfonic acid buffer solution.
  • the other steps were the same as in Example 1 to obtain a dialysis membrane.
  • Example 2 the 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 6 mg/mL lysozyme 4-hydroxyethylpiperazineethanesulfonic acid buffer solution.
  • the other steps were the same as in Example 1 to obtain a dialysis membrane.
  • Example 2 the 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 8 mg/mL lysozyme 4-hydroxyethylpiperazineethanesulfonic acid buffer solution.
  • the other steps were the same as in Example 1 to obtain a dialysis membrane.
  • Example 1 the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 10 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution.
  • the other steps were the same as in Example 1 to obtain a dialysis membrane.
  • Example 2 the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 20 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution.
  • the other steps were the same as in Example 1 to obtain a dialysis membrane.
  • Example 1 the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 30 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution.
  • the other steps were the same as in Example 1 to obtain a dialysis membrane.
  • the inventors characterized the crosslinked lysozyme nanofilms prepared in Examples 1 to 7 by scanning electron microscopy, and the results showed that the corresponding film thicknesses were 50 (see Fig. 1), 60, 90, 100, 120, 220 and 250 nm, respectively.
  • the nano-film is formed by densely stacking 20 nm-sized oligomers after lysozyme phase transition (see Figures 2 and 3).
  • the prepared cross-linked lysozyme nano film was controlled with a pore size of 6 mm PET, and the film was floated in 5 mL of ultrapure water, and 50 ⁇ L of 0.5 mg/mL PEG of different molecular weight was added dropwise to the film.
  • the UV-visible absorption spectrum was used to monitor the permeability of the solution before and after standing for 24 hours, and the pore size distribution of the membrane was analyzed. The results are shown in Fig. 4.
  • the pore size distribution of the membrane was obtained by fitting the membrane to the rejection of different molecular weights, and the pore size of the membrane decreased from 3.4 nm to 1.8 nm as the membrane thickness increased.
  • the dialysis membrane of Example 1 is used for separating methyl blue and methyl orange, methyl blue and rhodamine B, myoglobin and insulin, bovine serum albumin and insulin, and the specific method is as follows:
  • the dialysis membrane was floated in 5 mL of ultrapure water, and 50 ⁇ L of an aqueous solution containing 50 mg/L methyl blue and 50 mg/L methyl orange, 50 ⁇ L of 50 mg/L methyl blue and 50 mg/L rhodamine were added dropwise to the membrane.
  • Aqueous solution 50 ⁇ L of aqueous solution containing 10 g/L myoglobin and 10 g/L insulin, 50 ⁇ L of aqueous solution containing 10 g/L myoglobin and 10 g/L insulin, 50 ⁇ L of aqueous solution containing 10 g/L bovine serum albumin and 10 g/L insulin After standing at room temperature for 24 hours, the solution was monitored for permeability by ultraviolet-visible absorption spectroscopy.
  • the experimental results show that the dialysis membrane can completely pass through molecules with a molecular diameter of less than 2 nm, the molecular diameter of more than 3 nm and the negatively charged methyl blue, bovine serum albumin and myoglobin can be completely retained, while the retention rate of methyl orange Only 0.3%, the rejection rate of rhodamine B was 1.4%, and the retention rate of insulin was 22.6%.
  • the diffusion rate of methyl orange reached 606 nmol cm -2 h -1 , and the diffusion rate of rhodamine B was 308 nmol cm. -2 h -1 enables rapid and efficient separation of methyl blue and methyl orange, methyl blue and rhodamine, myoglobin and insulin, bovine serum albumin and insulin.
  • the dialysis membrane of Example 1 is used for removing urea, creatine liver, ⁇ -microglobulin and sulphate.
  • the specific method is as follows:
  • the simulated liquid is an aqueous solution containing 1 mg/mL bovine serum albumin and 25 mg/L sulphuric acid phenol, 40 mg/L ⁇ -microglobulin, 100 mg/L creatine liver and 1.5 mg/mL urea, simulating solution and dialysate (water The flow rate was 10 mL/min, and dialyzed against a dialysis membrane for 4 hours. The dialysis membrane was tested for the clearance rate of bovine serum albumin and sulphate, ⁇ -microglobulin, creatine liver and urea. As shown in Fig.
  • the dialysis membrane was able to retain macromolecular bovine serum albumin, The clearance effects of urea, ⁇ -microglobulin and creatine liver were better, and the clearance rates were 33.1%, 82.2%, 50.3% and 81.3%, respectively.
  • Nano-film the two-dimensional nano-film of bovine serum albumin at the gas-liquid interface was transferred to a solution of genipin in a mass fraction of 1%, and after cross-linking at room temperature for 2 hours, a cross-linked bovine serum albumin nano-film was obtained.
  • the obtained cross-linked bovine serum albumin nano-film was adhered to a PC film with a membrane diameter of 25 mm, a thickness of 12 ⁇ m and a pore size of 10 ⁇ m, and the cross-linked bovine serum albumin nano-film was used as a dense skin layer and the PC film was used as a support layer. Dialysis membrane.
  • the application of the dialysis membrane in removing urea, creatine liver, ⁇ -microglobulin, and indoxyl sulfate was the same as in Example 9.
  • the experimental results show that the dialysis membrane can retain the macromolecular bovine serum albumin, and the scavenging effect on sulphate, urea, ⁇ -microglobulin and creatine liver is better, and the clearance rate is 44.2%, 90.2%, 60.4%. And 88.6%.
  • 60 ⁇ L of 50 mmol/L tris(2-carboxyethyl)phosphine 4-hydroxyethylpiperazineethanesulfonic acid buffer solution was adjusted to pH 4.0 with NaOH, and then it was combined with 60 ⁇ L of 2 mg/mL ⁇ -lactalbumin 4 -Hydroxyethylpiperazineethanesulfonic acid buffer solution was evenly mixed and directly spread on the surface of 18mm ⁇ 18mm coverslips, and incubated at room temperature for 2 hours to form a layer of ⁇ -lactalbumin two-dimensional on the liquid-liquid interface of the cover glass.
  • the obtained cross-linked ⁇ -lactal albumin nano film was adhered to a PP film with a diameter of 25 mm, a thickness of 12 ⁇ m and a pore size of 10 ⁇ m to obtain a crosslinked ⁇ -lactal albumin nano film as a dense skin layer and a PP film as a support layer.
  • Dialysis membrane Dialysis membrane.
  • the application of the dialysis membrane in removing urea, creatine liver, ⁇ -microglobulin, and indoxyl sulfate was the same as in Example 9.
  • the experimental results show that the dialysis membrane has the same application as in the removal of urea, creatine liver, ⁇ -microglobulin and sulphate.
  • the dialysis membrane can completely retain the macromolecular bovine serum albumin against barium sulfate.
  • the scavenging effects of phenol, urea, ⁇ -microglobulin and creatine liver were better, and the clearance rates were 35.1%, 89.2%, 51.4% and 87.5%, respectively.
  • Insulin nanofilm Insulin nanofilm.
  • the obtained crosslinked insulin nanofilm was adhered to a PSF film having a membrane diameter of 25 mm, a thickness of 12 ⁇ m, and a pore size of 10 ⁇ m to obtain a dialysis membrane having a crosslinked insulin nanofilm as a dense skin layer and a PSF film as a support layer.
  • the application of the dialysis membrane in removing urea, creatine liver, ⁇ -microglobulin, and indoxyl sulfate was the same as in Example 9.
  • the experimental results show that the dialysis membrane has the same application as in the removal of urea, creatine liver, ⁇ -microglobulin and sulphate.
  • the dialysis membrane can completely retain the macromolecular bovine serum albumin against barium sulfate.
  • the scavenging effects of phenol, urea, ⁇ -microglobulin and creatine liver were better, and the clearance rates were 32.1%, 83.6%, 50.1% and 86.1%, respectively.

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  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Manufacturing & Machinery (AREA)
  • Health & Medical Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Urology & Nephrology (AREA)
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Abstract

A crosslinked protein-based separation membrane and an application thereof. The crosslinked protein-based separation membrane is a separation membrane formed by crosslinking, using a crosslinker, a two-dimensional nanofilm formed by phase transition of protein and then attaching the crosslinked two-dimensional nanofilm to a porous membrane, wherein the separation film has the crosslinked protein nanofilm as a compact surface layer and the porous membrane as a support layer. The protein is selected from any one of lysozyme, bovine serum albumin, insulin, and α-lactalbumin. The crosslinked protein-based separation membrane has good biocompatibility, can be used as a dialysis membrane for blood purification, and has a high retention rate for macromolecular proteins.

Description

一种基于交联蛋白质的分离膜及其应用Separation membrane based on crosslinked protein and application thereof 技术领域Technical field
本发明涉及一种分离膜，具体涉及一种制备简单、绿色、温和且孔径可控的分离膜，以及该分离膜的应用。The present invention relates to a separation membrane, and in particular to a separation membrane which is simple to prepare, green, mild and controllable in pore size, and the use of the separation membrane.
背景技术Background technique
膜分离技术由于具有分离效率高、无二次污染及操作简单等特点，目前已广泛运用于石油化工、污水处理、医药卫生、食品加工等领域。膜材料是膜分离技术的核心内容之一，目前常用的膜材料主要为有机合成高分子材料，然而聚合物膜的制备方法复杂，通常容易吸附原料液中的有机物质（如蛋白质、胶体和微生物等）；在与血液接触时，膜表面的蛋白质非特异性吸附还会引起凝血等负面效应，造成分离膜的渗透量降低，分离特异性降低。血液透析治疗是目前***患者常用的治疗手段之一，血液净化膜主要通过扩散/对流原理进行物质交换，并达到清除体内的代谢废物，维持电解质和清除体内过多的水分为目的。而常规血液透析膜无法达到对中大分子毒素的清除。因此，开发具有良好分离性能的同时，还具有简单易行，价格低廉的分离膜具有重要的现实意义。Membrane separation technology has been widely used in petrochemical, sewage treatment, medical and health, food processing and other fields due to its high separation efficiency, no secondary pollution and simple operation. Membrane material is one of the core contents of membrane separation technology. Currently, the commonly used membrane materials are mainly organic synthetic polymer materials. However, the preparation method of polymer membrane is complicated, and it is easy to adsorb organic substances (such as proteins, colloids and microorganisms in the raw material liquid). In the case of contact with blood, non-specific adsorption of proteins on the surface of the membrane may cause negative effects such as blood coagulation, resulting in a decrease in the amount of permeation of the separation membrane and a decrease in the specificity of separation. Hemodialysis treatment is one of the commonly used treatments for uremic patients. The blood purification membrane mainly exchanges substances through diffusion/convection principle, and achieves the purpose of eliminating metabolic wastes in the body, maintaining electrolytes and removing excess water from the body. Conventional hemodialysis membranes are unable to achieve the elimination of macromolecular toxins. Therefore, it is of great practical significance to develop a separation membrane which has good separation performance and is simple and inexpensive, and is inexpensive.
利用纳米结构材料自组装制备的选择性分离膜是近年来分离膜领域的一个新的尝试。时至今日，对于工业应用来说，具有选择透过性的大部分纳米复合膜并不能大面积生产。Selective separation membranes prepared by self-assembly of nanostructured materials are a new attempt in the field of separation membranes in recent years. Today, for industrial applications, most nanocomposite membranes with selective permeability are not produced on a large scale.
技术问题technical problem
本发明所要解决的技术问题在于针对上述现有技术的缺陷，提供一种基于交联蛋白质的分离膜，以及该分离膜的应用。The technical problem to be solved by the present invention is to provide a separation membrane based on crosslinked proteins and the use of the separation membrane in view of the above-mentioned drawbacks of the prior art.
技术解决方案Technical solution
解决上述技术问题所采用的基于交联蛋白质的分离膜是：将蛋白质相转变形成的蛋白质二维纳米薄膜用交联剂交联后粘附在多孔膜上，形成以交联蛋白质纳米薄膜为致密皮层、多孔膜为支撑层的分离膜。The cross-linked protein-based separation membrane used to solve the above technical problem is: a protein two-dimensional nano-film formed by phase-transformation of a protein is cross-linked with a crosslinking agent and adhered to the porous membrane to form a cross-linked protein nano-film as a dense The cortex and the porous membrane are separation membranes of the support layer.
上述的蛋白质为溶菌酶、牛血清白蛋白、胰岛素、α-乳白蛋白中任意一种。The above protein is any one of lysozyme, bovine serum albumin, insulin, and α-lactalbumin.
上述的蛋白质为溶菌酶时，溶菌酶相转变形成溶菌酶二维纳米薄膜的方法为：将10～100mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为6.0～8.0，然后将其与1～30mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液等体积混合后直接铺满基材表面，室温培育2～6小时，在基材上液体的气液界面形成一层溶菌酶二维纳米薄膜。When the above protein is lysozyme, the lysozyme phase is transformed into a two-dimensional nanofilm of lysozyme: buffering 4-hydroxyethylpiperazineethanesulfonic acid of 10-100 mmol/L tris(2-carboxyethyl)phosphine The solution is adjusted to pH 6.0-8.0 with NaOH, and then mixed with 1 to 30 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution in equal volume and directly spread on the surface of the substrate, and cultured at room temperature 2 ~6 hours, a layer of lysozyme two-dimensional nano-film was formed on the gas-liquid interface of the liquid on the substrate.
上述的蛋白质为牛血清白蛋白、胰岛素、α-乳白蛋白中任意一种时，蛋白质相转变形成的二维纳米薄膜的方法为：将10～100mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为4.0～6.0，然后将其与1～30mg/mL蛋白质的4-羟乙基哌嗪乙磺酸缓冲溶液等体积混合后直接铺满基材表面，室温培育2～6小时，在基材上液体的气液界面形成一层蛋白质二维纳米薄膜。When the above protein is any one of bovine serum albumin, insulin and α-lactalbumin, the two-dimensional nanofilm formed by protein phase transformation is: 10 to 100 mmol/L of tris(2-carboxyethyl)phosphine The 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution was adjusted to a pH of 4.0 to 6.0 with NaOH, and then mixed with an equal volume of 1-hydroxyethylpiperazineethanesulfonic acid buffer solution of 1 to 30 mg/mL protein. The surface of the substrate is directly spread and incubated at room temperature for 2 to 6 hours to form a layer of protein two-dimensional nano-film on the liquid-liquid interface of the liquid on the substrate.
上述蛋白质相转变形成的蛋白质二维纳米薄膜用交联剂交联的方法为：将蛋白质二维纳米薄膜转移至质量分数为0.2%～2%的交联剂水溶液中，室温交联2～6小时。其中所述的交联剂为戊二醛、京尼平、谷氨酰胺转氨酶、碳二亚胺中任意一种。The protein two-dimensional nano film formed by the phase transition of the above protein is crosslinked by a crosslinking agent: the protein two-dimensional nano film is transferred to an aqueous solution of a crosslinking agent having a mass fraction of 0.2% to 2%, and crosslinked at room temperature by 2 to 6 hour. The crosslinking agent is any one of glutaraldehyde, genipin, glutamine transaminase, and carbodiimide.
上述的多孔膜为PET核孔膜、PC膜、PP膜、PSF膜、PTFE膜中任意一种，所述多孔膜的孔径大小为0.22～10μm。The porous film described above is any one of a PET nucleus membrane, a PC membrane, a PP membrane, a PSF membrane, and a PTFE membrane, and the pore size of the porous membrane is 0.22 to 10 μm.
本发明基于交联蛋白质的分离膜可作为透析膜或超滤膜使用。The separation membrane based on the crosslinked protein of the present invention can be used as a dialysis membrane or an ultrafiltration membrane.
本发明基于交联蛋白质的分离膜作为透析膜在混合蛋白质分离中的应用，其中所述的混合蛋白质如牛血清蛋白和胰岛素，或者肌红蛋白和胰岛素。The present invention is based on the use of a separation membrane of crosslinked proteins as a dialysis membrane for the separation of mixed proteins such as bovine serum albumin and insulin, or myoglobin and insulin.
本发明基于交联蛋白质的分离膜作为透析膜在混合染料分离中的应用，其中所述的混合染料如甲基蓝和甲基橙，或者甲基蓝和罗丹明B。The present invention is based on the use of a crosslinked protein separation membrane as a dialysis membrane for the separation of mixed dyes such as methyl blue and methyl orange, or methyl blue and rhodamine B.
本发明基于交联蛋白质的分离膜作为透析膜在血液透析去除尿毒素中的应用，其中所述的尿毒素为相对分子量<500的毒素，如尿素、肌酸肝等，以及相对分子量为500～20000的中分子量毒素，如β-微球蛋白、甲状旁腺激素、瘦素、肾素、蛋白质结合毒素硫酸吲哚酚等。The invention is based on the use of a separation membrane of crosslinked protein as a dialysis membrane for removing urinary toxins from hemodialysis, wherein the urinary toxin is a toxin having a relative molecular weight of <500, such as urea, creatine, etc., and a relative molecular weight of 500 ～. Medium molecular weight toxins of 20000, such as β-microglobulin, parathyroid hormone, leptin, renin, protein-binding toxin, sulphuric acid, and the like.
有益效果Beneficial effect
1、本发明分离膜由蛋白质的寡聚物密堆积而成，无色透明，为纯蛋白质膜，根据蛋白质的浓度可以控制膜的厚度，且随膜厚度的增加其孔大小从3.4nm减小至1.8nm。1. The separation membrane of the present invention is densely packed from oligomers of proteins, is colorless and transparent, and is a pure protein membrane. The thickness of the membrane can be controlled according to the concentration of the protein, and the pore size decreases from 3.4 nm as the thickness of the membrane increases. To 1.8nm.
2、本发明分离膜具有较好的生物相容性，可作为透析膜用于血液净化，能够清除尿素、肌酸肝及中大分子毒素和硫酸吲哚酚等，且对于大分子蛋白具有更高的截留率，是一种较为理想的透析膜材料。2. The separation membrane of the invention has good biocompatibility, can be used as a dialysis membrane for blood purification, can remove urea, creatine liver and medium macrotoxin and sulphuric acid phenol, and has more for macromolecular proteins. High rejection rate is an ideal dialysis membrane material.
3、本发明分离膜能够有效分离不同大小的混合蛋白质以及不同大小的混合染料，如牛血清蛋白和胰岛素、肌红蛋白和胰岛素、甲基蓝和甲基橙、甲基蓝和罗丹明B。3. The separation membrane of the present invention is capable of efficiently separating mixed proteins of different sizes and mixed dyes of different sizes, such as bovine serum albumin and insulin, myoglobin and insulin, methyl blue and methyl orange, methyl blue and rhodamine B.
4、本发明分离膜制备方法简单，易实现大面积制备，同时又具有低成本、低能耗、环保等特点，避免了传统聚合物膜合成过程中的步骤繁琐及环境污染问题。4. The preparation method of the separation membrane of the invention is simple, easy to realize large-area preparation, and has the characteristics of low cost, low energy consumption, environmental protection, etc., avoiding the cumbersome steps and environmental pollution problems in the traditional polymer membrane synthesis process.
附图说明DRAWINGS
图1是实施例1中交联溶菌酶纳米薄膜的扫描电镜图。1 is a scanning electron micrograph of a crosslinked lysozyme nanofilm of Example 1.
图2是实施例1中交联溶菌酶纳米薄膜的透射电镜平面图。2 is a transmission electron microscope plan view of the crosslinked lysozyme nanofilm of Example 1.
图3是实施例1中交联溶菌酶纳米薄膜的原子力显微镜图。3 is an atomic force microscope chart of the crosslinked lysozyme nanofilm of Example 1.
图4是不同厚度的交联溶菌酶纳米薄膜的孔径分布。Figure 4 is a pore size distribution of crosslinked lysozyme nanofilms of different thicknesses.
图5是实施例1得到的透析膜对尿素、硫酸吲哚酚、肌酸肝及β-微球蛋白的清除。Fig. 5 is a view showing the removal of urea, sulphate, creatine and β-microglobulin by the dialysis membrane obtained in Example 1.
本发明的最佳实施方式BEST MODE FOR CARRYING OUT THE INVENTION
实施例1Example 1
将60μL 50mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为7.0，然后将其与60μL 2mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液混合均匀后直接铺满在18mm×18mm的盖玻片表面，室温培育2小时，在盖玻片上液体的气液界面形成一层溶菌酶二维纳米薄膜；将气液界面的溶菌酶二维纳米薄膜转移至质量分数为1%的戊二醛水溶液中，室温交联2小时后，得到交联溶菌酶纳米薄膜。60 μL of 50 mmol/L tris(2-carboxyethyl)phosphine 4-hydroxyethylpiperazineethanesulfonic acid buffer solution was adjusted to pH 7.0 with NaOH, and then combined with 60 μL of 2 mg/mL lysozyme 4-hydroxyl The ethyl piperazine ethanesulfonic acid buffer solution was uniformly mixed and directly spread on the surface of the 18 mm × 18 mm coverslip, and incubated at room temperature for 2 hours to form a layer of lysozyme two-dimensional nano-film on the liquid-liquid interface of the cover glass. The lysozyme two-dimensional nanofilm at the gas-liquid interface was transferred to an aqueous solution of glutaraldehyde having a mass fraction of 1%, and after cross-linking at room temperature for 2 hours, a cross-linked lysozyme nanofilm was obtained.
将上述交联溶菌酶纳米薄膜粘附在膜直径为25mm、厚度为12μm、孔径大小为10μm的PET核孔膜上，得到以交联溶菌酶纳米薄膜为致密皮层、PET核孔膜为支撑层的透析膜。The cross-linked lysozyme nano-film was adhered to a PET nuclear pore membrane with a membrane diameter of 25 mm, a thickness of 12 μm and a pore size of 10 μm, and a cross-linked lysozyme nanofilm was used as a dense skin layer and a PET nucleus membrane as a support layer. Dialysis membrane.
本发明的实施方式Embodiments of the invention
实施例2Example 2
本实施例中，用等体积 4mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液替换实施例1中2mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液，其他步骤与实施例1相同，得到透析膜。In this example, the 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 4 mg/mL lysozyme 4-hydroxyethylpiperazineethanesulfonic acid buffer solution. The other steps were the same as in Example 1 to obtain a dialysis membrane.
实施例3Example 3
本实施例中，用等体积 6mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液替换实施例1中2mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液，其他步骤与实施例1相同，得到透析膜。In this example, the 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 6 mg/mL lysozyme 4-hydroxyethylpiperazineethanesulfonic acid buffer solution. The other steps were the same as in Example 1 to obtain a dialysis membrane.
实施例4Example 4
本实施例中，用等体积 8mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液替换实施例1中2mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液，其他步骤与实施例1相同，得到透析膜。In this example, the 4-hydroxyethylpiperazineethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 8 mg/mL lysozyme 4-hydroxyethylpiperazineethanesulfonic acid buffer solution. The other steps were the same as in Example 1 to obtain a dialysis membrane.
实施例5Example 5
本实施例中，用等体积 10mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液替换实施例1中2mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液，其他步骤与实施例1相同，得到透析膜。In this embodiment, the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 10 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution. The other steps were the same as in Example 1 to obtain a dialysis membrane.
实施例6Example 6
本实施例中，用等体积 20mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液替换实施例1中2mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液，其他步骤与实施例1相同，得到透析膜。In this example, the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 20 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution. The other steps were the same as in Example 1 to obtain a dialysis membrane.
实施例7Example 7
本实施例中，用等体积 30mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液替换实施例1中2mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液，其他步骤与实施例1相同，得到透析膜。In this embodiment, the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of 2 mg/mL lysozyme in Example 1 was replaced with an equal volume of 30 mg/mL lysozyme 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution. The other steps were the same as in Example 1 to obtain a dialysis membrane.
发明人对实施例1～7中制备的交联溶菌酶纳米薄膜进行扫描电镜表征，结果显示其对应的膜厚度依次为50（见图1）、60、90、100、120、220和250nm，该纳米薄膜是由溶菌酶相转变后的20nm大小的寡聚物密堆积而成（如图2、3）。将制备的交联溶菌酶纳米薄膜用孔径为6mm的PET对其面积进行控制，将该膜漂浮于5mL超纯水中，且在该膜上滴加50μL 0.5mg/mL不同分子量的PEG，采用紫外可见吸收光谱对静置24小时前后的溶液进行透过性监控，并分析膜的孔径大小分布，结果如图4所示。通过拟合膜对不同分子量的截留率得到膜的孔径大小分布，随着膜厚度的增加膜的孔径从3.4nm减小至1.8nm。The inventors characterized the crosslinked lysozyme nanofilms prepared in Examples 1 to 7 by scanning electron microscopy, and the results showed that the corresponding film thicknesses were 50 (see Fig. 1), 60, 90, 100, 120, 220 and 250 nm, respectively. The nano-film is formed by densely stacking 20 nm-sized oligomers after lysozyme phase transition (see Figures 2 and 3). The prepared cross-linked lysozyme nano film was controlled with a pore size of 6 mm PET, and the film was floated in 5 mL of ultrapure water, and 50 μL of 0.5 mg/mL PEG of different molecular weight was added dropwise to the film. The UV-visible absorption spectrum was used to monitor the permeability of the solution before and after standing for 24 hours, and the pore size distribution of the membrane was analyzed. The results are shown in Fig. 4. The pore size distribution of the membrane was obtained by fitting the membrane to the rejection of different molecular weights, and the pore size of the membrane decreased from 3.4 nm to 1.8 nm as the membrane thickness increased.
实施例8Example 8
实施例1的透析膜在分离甲基蓝和甲基橙、甲基蓝和罗丹明B、肌红蛋白和胰岛素、牛血清蛋白和胰岛素中的应用，具体方法如下：The dialysis membrane of Example 1 is used for separating methyl blue and methyl orange, methyl blue and rhodamine B, myoglobin and insulin, bovine serum albumin and insulin, and the specific method is as follows:
将透析膜漂浮于5mL超纯水中，分别在该膜上滴加50μL含50mg/L甲基蓝和50mg/L甲基橙的水溶液、50μL含50mg/L甲基蓝和50mg/L罗丹明的水溶液、50μL含10g/L肌红蛋白和10g/L胰岛素的水溶液、50μL含10g/L肌红蛋白和10g/L胰岛素的水溶液、50μL含10g/L牛血清蛋白和10g/L胰岛素的水溶液，室温静置24小时后，采用紫外可见吸收光谱对溶液进行透过性监控。实验结果显示，该透析膜能够完全通过分子直径小于2nm的分子，分子直径大于3nm并且带有负电荷的甲基蓝、牛血清蛋白和肌红蛋白能够被完全截留，而甲基橙的截留率仅为0.3%、罗丹明B的截留率为1.4%、胰岛素的截留率为22.6%，其中甲基橙的扩散速率达到606 nmol cm -2 h -1，罗丹明B的扩散速率为308 nmol cm -2 h -1，能够实现甲基蓝和甲基橙、甲基蓝和罗丹明、肌红蛋白和胰岛素、牛血清蛋白和胰岛素的快速、有效分离。 The dialysis membrane was floated in 5 mL of ultrapure water, and 50 μL of an aqueous solution containing 50 mg/L methyl blue and 50 mg/L methyl orange, 50 μL of 50 mg/L methyl blue and 50 mg/L rhodamine were added dropwise to the membrane. Aqueous solution, 50 μL of aqueous solution containing 10 g/L myoglobin and 10 g/L insulin, 50 μL of aqueous solution containing 10 g/L myoglobin and 10 g/L insulin, 50 μL of aqueous solution containing 10 g/L bovine serum albumin and 10 g/L insulin After standing at room temperature for 24 hours, the solution was monitored for permeability by ultraviolet-visible absorption spectroscopy. The experimental results show that the dialysis membrane can completely pass through molecules with a molecular diameter of less than 2 nm, the molecular diameter of more than 3 nm and the negatively charged methyl blue, bovine serum albumin and myoglobin can be completely retained, while the retention rate of methyl orange Only 0.3%, the rejection rate of rhodamine B was 1.4%, and the retention rate of insulin was 22.6%. The diffusion rate of methyl orange reached 606 nmol cm -2 h -1 , and the diffusion rate of rhodamine B was 308 nmol cm. -2 h -1 enables rapid and efficient separation of methyl blue and methyl orange, methyl blue and rhodamine, myoglobin and insulin, bovine serum albumin and insulin.
实施例9Example 9
实施例1的透析膜在去除尿素、肌酸肝、β-微球蛋白以及硫酸吲哚酚中的应用，具体方法如下：The dialysis membrane of Example 1 is used for removing urea, creatine liver, β-microglobulin and sulphate. The specific method is as follows:
模拟液是含有1mg/mL牛血清蛋白和25mg/L硫酸吲哚酚、40mg/L β-微球蛋白、100mg/L肌酸肝和1.5mg/mL尿素的水溶液，模拟液及透析液（水）的流速都为10mL/min，用透析膜透析4小时。测试透析膜对牛血清蛋白和硫酸吲哚酚、β-微球蛋白、肌酸肝、尿素的清除率，如图5所示，该透析膜能够截留大分子牛血清蛋白，对硫酸吲哚酚、尿素、β-微球蛋白及肌酸肝的清除效果较好，清除率依次为33.1%、82.2%、50.3%和81.3%。The simulated liquid is an aqueous solution containing 1 mg/mL bovine serum albumin and 25 mg/L sulphuric acid phenol, 40 mg/L β-microglobulin, 100 mg/L creatine liver and 1.5 mg/mL urea, simulating solution and dialysate (water The flow rate was 10 mL/min, and dialyzed against a dialysis membrane for 4 hours. The dialysis membrane was tested for the clearance rate of bovine serum albumin and sulphate, β-microglobulin, creatine liver and urea. As shown in Fig. 5, the dialysis membrane was able to retain macromolecular bovine serum albumin, The clearance effects of urea, β-microglobulin and creatine liver were better, and the clearance rates were 33.1%, 82.2%, 50.3% and 81.3%, respectively.
实施例10Example 10
将60μL 50mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为5.0，然后将其与60μL 5mg/mL牛血清白蛋白的4-羟乙基哌嗪乙磺酸缓冲溶液混合均匀后直接铺满在18mm×18mm的盖玻片表面，室温培育2小时，在盖玻片上液体的气液界面形成一层牛血清白蛋白二维纳米薄膜；将气液界面的牛血清白蛋白二维纳米薄膜转移至质量分数为1%的京尼平水溶液中，室温交联2小时后，得到交联牛血清白蛋白纳米薄膜。将所得交联牛血清白蛋白纳米薄膜粘附在膜直径为25mm、厚度为12μm、孔径大小为10μm的PC膜上，得到以交联牛血清白蛋白纳米薄膜为致密皮层、PC膜为支撑层的透析膜。该透析膜在去除尿素、肌酸肝、β-微球蛋白以及硫酸吲哚酚中的应用与实施例9相同。实验结果显示，该透析膜能够截留大分子牛血清蛋白，对硫酸吲哚酚、尿素、β-微球蛋白及肌酸肝的清除效果较好，清除率依次为44.2%、90.2%、60.4%和88.6%。60 μL of 50 mmol/L tris(2-carboxyethyl)phosphine 4-hydroxyethylpiperazineethanesulfonic acid buffer solution was adjusted to pH 5.0 with NaOH, and then it was combined with 60 μL of 5 mg/mL bovine serum albumin 4 -Hydroxyethylpiperazine ethanesulfonic acid buffer solution was evenly mixed and directly spread on the surface of 18mm × 18mm coverslips, incubated at room temperature for 2 hours, forming a layer of bovine serum albumin two-dimensional on the liquid-liquid interface of the cover glass. Nano-film; the two-dimensional nano-film of bovine serum albumin at the gas-liquid interface was transferred to a solution of genipin in a mass fraction of 1%, and after cross-linking at room temperature for 2 hours, a cross-linked bovine serum albumin nano-film was obtained. The obtained cross-linked bovine serum albumin nano-film was adhered to a PC film with a membrane diameter of 25 mm, a thickness of 12 μm and a pore size of 10 μm, and the cross-linked bovine serum albumin nano-film was used as a dense skin layer and the PC film was used as a support layer. Dialysis membrane. The application of the dialysis membrane in removing urea, creatine liver, β-microglobulin, and indoxyl sulfate was the same as in Example 9. The experimental results show that the dialysis membrane can retain the macromolecular bovine serum albumin, and the scavenging effect on sulphate, urea, β-microglobulin and creatine liver is better, and the clearance rate is 44.2%, 90.2%, 60.4%. And 88.6%.
实施例11Example 11
将60μL 50mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为4.0，然后将其与60μL 2mg/mL α-乳白蛋白的4-羟乙基哌嗪乙磺酸缓冲溶液混合均匀后直接铺满在18mm×18mm的盖玻片表面，室温培育2小时，在盖玻片上液体的气液界面形成一层α-乳白蛋白二维纳米薄膜；将气液界面的α-乳白蛋白二维纳米薄膜转移至质量分数为1%的谷氨酰胺转氨酶水溶液中，室温交联2小时后，得到交联α-乳白蛋白纳米薄膜。将所得交联α-乳白蛋白纳米薄膜粘附在膜直径为25mm、厚度为12μm、孔径大小为10μm的PP膜上，得到以交联α-乳白蛋白纳米薄膜为致密皮层、PP膜为支撑层的透析膜。该透析膜在去除尿素、肌酸肝、β-微球蛋白以及硫酸吲哚酚中的应用与实施例9相同。实验结果显示，该透析膜在去除尿素、肌酸肝、β-微球蛋白以及硫酸吲哚酚中的应用与实施例9相同，该透析膜能够完全截留大分子牛血清蛋白，对硫酸吲哚酚、尿素、β-微球蛋白及肌酸肝的清除效果较好，清除率依次为35.1%、89.2%、51.4%和87.5%。60 μL of 50 mmol/L tris(2-carboxyethyl)phosphine 4-hydroxyethylpiperazineethanesulfonic acid buffer solution was adjusted to pH 4.0 with NaOH, and then it was combined with 60 μL of 2 mg/mL α-lactalbumin 4 -Hydroxyethylpiperazineethanesulfonic acid buffer solution was evenly mixed and directly spread on the surface of 18mm × 18mm coverslips, and incubated at room temperature for 2 hours to form a layer of α-lactalbumin two-dimensional on the liquid-liquid interface of the cover glass. Nano-film; the α-lactal albumin two-dimensional nano-film at the gas-liquid interface was transferred to a 1% by mass aqueous solution of glutamine transaminase, and after cross-linking at room temperature for 2 hours, a cross-linked α-lactalbumin nanofilm was obtained. The obtained cross-linked α-lactal albumin nano film was adhered to a PP film with a diameter of 25 mm, a thickness of 12 μm and a pore size of 10 μm to obtain a crosslinked α-lactal albumin nano film as a dense skin layer and a PP film as a support layer. Dialysis membrane. The application of the dialysis membrane in removing urea, creatine liver, β-microglobulin, and indoxyl sulfate was the same as in Example 9. The experimental results show that the dialysis membrane has the same application as in the removal of urea, creatine liver, β-microglobulin and sulphate. The dialysis membrane can completely retain the macromolecular bovine serum albumin against barium sulfate. The scavenging effects of phenol, urea, β-microglobulin and creatine liver were better, and the clearance rates were 35.1%, 89.2%, 51.4% and 87.5%, respectively.
实施例12Example 12
将60μL 50mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为6.0，然后将其与60μL 2mg/mL胰岛素的4-羟乙基哌嗪乙磺酸缓冲溶液混合均匀后直接铺满在18mm×18mm的盖玻片表面，室温培育2小时，在盖玻片上液体的气液界面形成一层胰岛素二维纳米薄膜；将气液界面的胰岛素二维纳米薄膜转移至质量分数为1%的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐水溶液中，室温交联2小时后，得到交联胰岛素纳米薄膜。将所得交联胰岛素纳米薄膜粘附在膜直径为25mm、厚度为12μm、孔径大小为10μm的PSF膜上，得到以交联胰岛素纳米薄膜为致密皮层、PSF膜为支撑层的透析膜。该透析膜在去除尿素、肌酸肝、β-微球蛋白以及硫酸吲哚酚中的应用与实施例9相同。实验结果显示，该透析膜在去除尿素、肌酸肝、β-微球蛋白以及硫酸吲哚酚中的应用与实施例9相同，该透析膜能够完全截留大分子牛血清蛋白，对硫酸吲哚酚、尿素、β-微球蛋白及肌酸肝的清除效果较好，清除率依次为32.1%、83.6%、50.1%和86.1%。60 μL of 50 mmol/L tris(2-carboxyethyl)phosphine 4-hydroxyethylpiperazineethanesulfonic acid buffer solution was adjusted to pH 6.0 with NaOH, and then it was combined with 60 μL of 2 mg/mL insulin 4-hydroxyethyl The piperazine ethanesulfonic acid buffer solution was uniformly mixed and directly spread on the surface of the 18 mm × 18 mm coverslip, and incubated at room temperature for 2 hours to form a layer of insulin two-dimensional nano-film on the liquid-liquid interface of the cover glass; The two-dimensional nano-film of insulin was transferred to an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a mass fraction of 1%, and crosslinked at room temperature for 2 hours to obtain cross-linking. Insulin nanofilm. The obtained crosslinked insulin nanofilm was adhered to a PSF film having a membrane diameter of 25 mm, a thickness of 12 μm, and a pore size of 10 μm to obtain a dialysis membrane having a crosslinked insulin nanofilm as a dense skin layer and a PSF film as a support layer. The application of the dialysis membrane in removing urea, creatine liver, β-microglobulin, and indoxyl sulfate was the same as in Example 9. The experimental results show that the dialysis membrane has the same application as in the removal of urea, creatine liver, β-microglobulin and sulphate. The dialysis membrane can completely retain the macromolecular bovine serum albumin against barium sulfate. The scavenging effects of phenol, urea, β-microglobulin and creatine liver were better, and the clearance rates were 32.1%, 83.6%, 50.1% and 86.1%, respectively.

Claims (10)

  1. 一种基于交联蛋白质的分离膜，其特征在于：该分离膜是蛋白质相转变形成的二维纳米薄膜用交联剂交联后粘附在多孔膜上，形成的以交联蛋白质纳米薄膜为致密皮层、多孔膜为支撑层的分离膜；A separation membrane based on cross-linked protein, characterized in that the separation membrane is a two-dimensional nano-film formed by phase transformation of a protein, which is cross-linked with a crosslinking agent and adhered to the porous membrane to form a cross-linked protein nano-film. a dense skin layer, a porous membrane is a separation membrane of the support layer;
    上述的蛋白质为溶菌酶、牛血清白蛋白、胰岛素、α-乳白蛋白中任意一种。The above protein is any one of lysozyme, bovine serum albumin, insulin, and α-lactalbumin.
  2. 根据权利要求1所述的基于交联蛋白质的分离膜，其特征在于：所述的蛋白质为溶菌酶时，溶菌酶相转变形成溶菌酶二维纳米薄膜的方法为：将10～100mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为6.0～8.0，然后将其与1～30mg/mL溶菌酶的4-羟乙基哌嗪乙磺酸缓冲溶液等体积混合后直接铺满基材表面，室温培育2～6小时，在基材上液体的气液界面形成一层溶菌酶二维纳米薄膜。The cross-linked protein-based separation membrane according to claim 1, wherein when the protein is lysozyme, the lysozyme phase is transformed into a lysozyme two-dimensional nano-film by: 10 to 100 mmol/L. 4-Hydroxyethylpiperazineethanesulfonic acid buffer solution of (2-carboxyethyl)phosphine was adjusted to pH 6.0-8.0 with NaOH, and then it was mixed with 1-hydroxyethylperidine of 1-3 mg/mL lysozyme. The oxalic acid sulfonic acid buffer solution is directly mixed with the surface of the substrate and incubated for 2 to 6 hours at room temperature to form a two-dimensional nano-film of lysozyme on the liquid-liquid interface of the liquid on the substrate.
  3. 根据权利要求1所述的基于交联蛋白质的分离膜，其特征在于：所述的蛋白质为牛血清白蛋白、胰岛素、α-乳白蛋白中任意一种时，所述蛋白质相转变形成的二维纳米薄膜的方法为：将10～100mmol/L三(2-羧乙基)膦的4-羟乙基哌嗪乙磺酸缓冲溶液用NaOH调节至pH值为4.0～6.0，然后将其与1～30mg/mL蛋白质的4-羟乙基哌嗪乙磺酸缓冲溶液等体积混合后直接铺满基材表面，室温培育2～6小时，在基材上液体的气液界面形成一层蛋白质二维纳米薄膜。The crosslinked protein-based separation membrane according to claim 1, wherein when the protein is any one of bovine serum albumin, insulin, and α-lactalbumin, the phase transition of the protein phase is two-dimensionally formed. The nano film is prepared by adjusting a buffer solution of 4-hydroxyethylpiperazine ethanesulfonic acid of 10 to 100 mmol/L of tris(2-carboxyethyl)phosphine with NaOH to a pH of 4.0 to 6.0, and then combining it with 1 ~30mg/mL protein 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution is mixed in equal volume and directly spread on the surface of the substrate, and incubated at room temperature for 2 to 6 hours to form a layer of protein on the liquid-liquid interface of the liquid on the substrate. Vitamin nano film.
  4. 根据权利要求1所述的基于交联蛋白质的分离膜，其特征在于所述蛋白质相转变形成的蛋白质二维纳米薄膜用交联剂交联的方法为：将蛋白质二维纳米薄膜转移至质量分数为0.2%～2%的交联剂水溶液中，室温交联2～6小时。The cross-linked protein-based separation membrane according to claim 1, wherein the protein two-phase nano-film formed by phase transformation of the protein is cross-linked by a crosslinking agent by transferring the protein two-dimensional nano-film to a mass fraction. It is crosslinked at room temperature for 2 to 6 hours in an aqueous solution of 0.2% to 2% of a crosslinking agent.
  5. 根据权利要求1或4所述的基于交联蛋白质的分离膜，其特征在于：所述的交联剂为戊二醛、京尼平、谷氨酰胺转氨酶、碳二亚胺中任意一种。The crosslinked protein-based separation membrane according to claim 1 or 4, wherein the crosslinking agent is any one of glutaraldehyde, genipin, glutamine transaminase, and carbodiimide.
  6. 根据权利要求1所述的基于交联蛋白质的分离膜，其特征在于：所述的多孔膜为PET核孔膜、PC膜、PP膜、PSF膜、PTFE膜中任意一种。The crosslinked protein-based separation membrane according to claim 1, wherein the porous membrane is any one of a PET nucleus membrane, a PC membrane, a PP membrane, a PSF membrane, and a PTFE membrane.
  7. 根据权利要求6所述的基于交联蛋白质的分离膜，其特征在于：所述多孔膜的孔径大小为0.22～10μm。The crosslinked protein-based separation membrane according to claim 6, wherein the porous membrane has a pore size of from 0.22 to 10 μm.
  8. 根据权利要求1～7任意一项所述的基于交联蛋白质的分离膜，其特征在于：所述的分离膜为透析膜或超滤膜。The crosslinked protein-based separation membrane according to any one of claims 1 to 7, wherein the separation membrane is a dialysis membrane or an ultrafiltration membrane.
  9. 权利要求1所述的基于交联蛋白质的分离膜作为透析膜在混合蛋白质分离中的应用。Use of the crosslinked protein-based separation membrane of claim 1 as a dialysis membrane for the separation of mixed proteins.
  10. 根据权利要求9所述的基于交联蛋白质的分离膜作为透析膜在混合蛋白质分离中的应用，其特征在于：所述的混合蛋白质为牛血清蛋白和胰岛素，或者肌红蛋白和胰岛素。The crosslinked protein-based separation membrane according to claim 9 as a dialysis membrane for use in mixed protein separation, characterized in that the mixed protein is bovine serum albumin and insulin, or myoglobin and insulin.
    11、权利要求1所述的基于交联蛋白质的分离膜作为透析膜在混合染料分离中的应用。11. Use of a crosslinked protein-based separation membrane according to claim 1 as a dialysis membrane for the separation of mixed dyes.
    12、根据权利要求11所述的基于交联蛋白质的分离膜作为透析膜在混合染料分离中的应用，其特征在于：所述的混合染料为甲基蓝和甲基橙，或者甲基蓝和罗丹明B。The use of a crosslinked protein-based separation membrane according to claim 11 as a dialysis membrane for the separation of mixed dyes, characterized in that the mixed dye is methyl blue and methyl orange, or methyl blue and Rodin Ming B.
    13、权利要求1所述的基于交联蛋白质的分离膜作为透析膜在去除尿毒素中的应用。13. Use of a crosslinked protein-based separation membrane according to claim 1 as a dialysis membrane for removing urinary toxin.
    14、权利要求13所述的基于交联蛋白质的分离膜作为透析膜在去除尿毒素中的应用，其特征在于：所述的尿毒素为尿素、肌酸肝、β-微球蛋白、甲状旁腺激素、瘦素、肾素以及硫酸吲哚酚。The use of the crosslinked protein-based separation membrane according to claim 13 as a dialysis membrane for removing urinary toxin, characterized in that the urinary toxin is urea, creatine liver, β-microglobulin, and parathyroid Gland hormone, leptin, renin and sulphate.
     
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CN105296977A (en) * 2015-10-29 2016-02-03 陕西师范大学 Method for performing metal electroless deposition based on lysozyme two-dimension nano-film
CN105475359A (en) * 2015-11-24 2016-04-13 陕西师范大学 Application of two-dimensional lysozyme nanometer film as antibacterial material
CN107174983A (en) * 2016-03-10 2017-09-19 中国科学院过程工程研究所 A kind of polysulfones dialysis membrane and its preparation method and application
CN108854599A (en) * 2018-05-14 2018-11-23 陕西师范大学 A kind of dialysis membrane and its application based on crosslinking lysozyme

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